New tools for determining the three dimensional structure of single macromolecules and macromolecular complexes are urgently needed. The goal of this R01 renewal proposal is to create a molecular microscope capable of imaging the proton density of, or mapping the locations of electron spin labels in, a single copy of a protein or macromolecular complex. We will develop a molecular microscope capable of scanned-probe nuclear magnetic resonance (NMR) imaging and electron spin resonance (ESR) imaging. Instrument development will be directed towards the study of native and spin-labeled macromolecular complexes bound to a lipid bilayer or located in a cell membrane. Our specific aims are: (1) To exploit magnetic-tipped cantilevers and force-gradient detection of magnetic resonance to observe electron spin resonance (ESR) from a single copy of a nitroxide-labeled protein and to observe nuclear magnetic resonance (NMR) from a few hundred protons in a biomolecule; (2) To understand and mitigate (via sample preparation, cantilever design, and spin modulation schemes) excess force and force- gradient noise experienced by ultrasensitive cantilevers near surfaces in vacuum at cryogenic temperatures and to learn to prolong spin coherence time by giving more perfect spin flips in magnetic resonance force microscopy; and (3) To develop time- domain Fourier image encoding to increase the imaging speed of scanned-probe magnetic resonance.